Human respiratory syncytial (RS) virus, an enveloped virus that contains a single negative-sense strand of genomic RNA, is an important agent of pediatric respiratory tract disease. Previously, complete cDNAs and complete nucleotide sequences were obtained for nine of the ten known viral mRNAs. Here, synthetic oligodeoxynucleotides were used to direct dideoxynucleotide sequencing of intergenic and flanking regions in the viral genome. The results confirmed the sequences of the gene termini obtained from the cDNAs, showing that the nine viral mRNAs, initiate with the conserved sequence 5 prime GGGGCAAAUA.A.U.. and terminate with the conserved sequences 5 prime ... AGUA.UA(N)1-4-polyA. Comparison of the intergenic and flanking sequences with the complete mRNA sequences established unambiguously the 3' to 5' order of the nine genes on the viral genome. Each gene was immediately followed (in genome-sense) by an oligo U tract of 4-7 residues that might direct synthesis of poly A tails of the mRNAs by a reiterative copying mechanism. The intergenic regions varied in length from 1 to 52 nucleotides and displayed no obvious sequence conservation except that in all cases the last nucleotide (in genome-sense) was an A residue.